The present invention relates to ligands to Tumor Necrosis Factor receptors (TNF-Rs) which inhibit the effect of TNF but not its binding to the TNF-Rs, as well as to ligands interacting with other receptors of the TNF/NGF family.
Tumor necrosis factor (TNF) is a pleiotropic cytokine, produced by a number of cell types, mainly by activated macrophages. It is one of the principal mediators of the immune and inflammatory response. Interest in its function has greatly increased, recently, in view of evidence of the involvement of TNF in the pathogenesis of a wide range of disease states, including endotoxin shock, cerebral malaria and graft-versus-host reaction. Since many of the effects of TNF are deleterious to the organism, it is of great interest to find ways of blocking its action on host cells. An evident target for such intervention are the molecules to which TNF has to bind in order to exert its effects, namely the TNF-Rs. These molecules exist not only in cell-bound, but also in soluble forms, consisting of the cleaved extra-cellular domains of the intact receptors (see Nophar et al., EMBO Journal, 9(10):3269-78, 1990). The soluble receptors maintain the ability to bind TNF, and thus have the ability to block its function by competition with surface receptors.
Another method of TNF inhibition based on the principle of competing with cell-bound molecules, is the use of antibodies recognizing TNF receptors and blocking the ligand binding.
The cell surface TNF-Rs are expressed in almost all cells of the body. The various effects of TNF, the cytotoxic, growth-promoting and others, are all signaled by the TNF receptors upon the binding of TNF to them. Two forms of these receptors, which differ in molecular size: 55 and 75 kilodaltons, have been described, and will be called herein p55 and p75 TNF-R, respectively. It should be noted, however, that there exist publications which refer to these receptors also as p60 and p80.
The TNF-Rs belong to a family of receptors which are involved in other critical biological processes. Examples of these receptors are the low affinity NGF receptor, which plays an important role in the regulation of growth and differentiation of nerve cells. Several other receptors are involved in the regulation of lymphocyte growth, such as CDw40 and some others. Another member of the family is the FAS receptor also called APO, a receptor which is involved in signaling for apoptosis and which, based on a study with mice deficient in its function, seems to play an important role in the etiology of a lupus-like disease. Herein, this family of receptors is called xe2x80x9cTNF/NGF receptor familyxe2x80x9d.
One of the most striking features of TNF compared to other cytokines, thought to contribute to the pathogenesis of several diseases, is its ability to elicit cell death. The cell-killing activity of TNF is thought to be induced by the p55 receptor. However, this p55 receptor activity can be assisted by the p75 receptor, through a yet unknown mechanism.
Parent application No. 07/524,263 and European Patent publications 398,327 and 412,486 disclose antibodies to the soluble TNF-Rs. These antibodies were found to recognize the soluble TNF-Rs and to inhibit the binding of TNF to the TNF-Rs on the cell surface. Monovalent F(ab) fragments blocked the effect of TNF, while intact antibodies were observed to mimic the cytotoxic effect of TNF. European patent publication 585,939 describes ligands interacting with a certain region in TNF-Rs.
The present invention provides a ligand to a member of the TNF/NGF receptor family, which binds either to the region of the fourth cysteine rich domain of such a receptor, or to the receptor between it and the cell membrane.
The region of the fourth cysteine rich domain will be called herein, for simplicity""s sake, the xe2x80x9c67 epitopexe2x80x9d and the antibodies recognizing it the xe2x80x9cgroup 67xe2x80x9d antibodies. This region may extend between about amino acids pro-141 and thr-179 in the p75 TNF-R (residues 163-201 of SEQ ID NO:2) or a corresponding region in another member of the TNF/NGF family. More particularly, the region may extend between about amino acids pro-141 and cys-163 of the p75 TNF-R (residues 163-185 of SEQ ID NO:2) or a corresponding region in another member of the TNF/NGF family. The ligand downstream of the fourth cysteine rich domain includes the amino acid sequence between about thr-179 and about the end of the extracellular domain of the receptor (residues 201-257 of SEQ ID NO:2) or a corresponding region in another member of the TNF/NGF family.
Preferably, the receptor is the TNF-R, in particular the p75 TNF-R.
One such ligand includes the amino acid sequence for the CDR region of the heavy chain of monoclonal antibody no. 67, and/or of the light chain thereof.
Another such ligand includes the amino acid sequence for the CDR region of the heavy chain of monoclonal antibody no. 81, and/or the light chain thereof.
Yet another such ligand includes the amino acid sequence or antibody against the xe2x80x9cstalkxe2x80x9d region, i.e., from about amino acid thr-181 to about amino acid 235-asp.
The ligands may comprise, for example, proteins, peptides, immunoadhesins, antibodies or other organic compounds.
The proteins may comprise, for example, a fusion protein of the ligand with another protein, optionally linked by a peptide linker. Such a fusion protein can increase the retention time of the ligand in the body, and thus may even allow the ligand-protein complex to be employed as a latent agent or as a vaccine.
The term xe2x80x9cproteinsxe2x80x9d includes muteins and fused proteins, their salts, functional derivatives and active fractions
xe2x80x9cFunctional derivativesxe2x80x9d as used herein cover derivatives of the ligands and their fused proteins and muteins, which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they cannot destroy the activity of the ligand and do not confer toxic properties on compositions containing it. These derivatives may, for example, include polyethylene glycol side-chains which may mask antigenic sites and extend the residence of the ligands in body fluids. Other derivatives include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed with acyl moieties (e.g., alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl groups (for example, that of seryl or threonyl residues) formed with acyl moieties.
As xe2x80x9cactive fractionsxe2x80x9d of the ligands, its fused proteins and its muteins, the present invention covers any fragment or precursors of the polypeptide chain of the ligand alone or together with associated molecules or residues linked thereto, e.g., sugar or phosphate residues, or aggregates of the protein molecule or the sugar residues by themselves, provided said fraction has the same biological and/or pharmaceutical activity.
As used herein the term xe2x80x9cmuteinsxe2x80x9d refers to analogs of the proteins, peptides and the like in which one or more or the amino acid residues of the protein found to bind are replaced by different amino acid residues or are deleted, or one or more amino acid residues are added to the original sequence, without changing considerably the activity of the resulting product. These muteins are prepared by known synthesis and/or by size-directed mutagenesis techniques, or any other known technique suitable therefor.
The term xe2x80x9cfused proteinxe2x80x9d refers to a polypeptide comprising the ligands or a mutein thereof fused with another protein which has an extended residence time in body fluids. The ligands may thus be fused to another protein, polypeptide or the like, e.g., an immunoglobulin or a fragment thereof.
The term xe2x80x9csaltsxe2x80x9d herein refers to both salts or carboxyl groups and to- acid addition salts of amino groups of the ligands, muteins and fused proteins thereof. Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids such as, for example, acetic acid or oxalic acid.
The peptides include peptide bond replacements and/or peptide mimetics, i.e., pseudopeptides, as known in the art (see, e.g., Proceedings of the 20th European Peptide Symposium, ed. G. Jung, E. Bayer, pp. 289-336, and references therein), as well as salts and pharmaceutical preparations and/or formulations which render the bioactive peptide(s) particularly suitable for oral, topical, nasal spray, ocular, pulmonary, I.V. or subcutaneous delivery, depending on the particular treatment indicated. Such salts, formulations, amino acid replacements and pseudopeptide structures may be necessary and desirable to enhance the stability, formulation, deliverability (e.g., slow release, prodrugs), or to improve the economy of production, as long as they do not adversely affect the biological activity of the peptide.
Besides substitutions, three particular forms of peptide mimetic and/or analogue structures of particular relevance when designating bioactive peptides, which have to bind to a receptor while risking the degradation by proteinases and peptidases in the blood, tissues and elsewhere, may be mentioned specifically, illustrated by the following examples: Firstly, the inversion of backbone chiral centres leading to D-amino acid residue structures may, particularly at the N-terminus, lead to enhanced stability for proteolytical degradation without adversely affecting activity. An example is given in the paper xe2x80x9cTritriated D-ala1-Peptide T Bindingxe2x80x9d, Smith C. S. et al., Drug Development Res. 15, pp. 371-379 (1988). Secondly, cyclic structure for stability, such as N to C interchain imides and lactams (Ede et al. in Smith and Rivier (Eds.) xe2x80x9cPeptides: Chemistry and Biology., Escom, Leiden (1991), pp. 268-270), and sometimes also receptor binding may be enhanced by forming cyclic analogues. An example of this is given in xe2x80x9cConfirmationally restricted thymopentin-like compoundsxe2x80x9d, U.S. Pat. No. 4,457,489 (1985), Goldstein, G. et al. Thirdly, the introduction of ketomethylene, methylsufide or retroinverse bonds to replace peptide bonds, i.e., the interchange of the CO and NH moieties are likely to enhance both stability and potency. An example of this type is given in the paper xe2x80x9cBiologically active retroinverso analogues of thymopentinxe2x80x9d, Sisto A. et al in Rivier, J. E. and Marshall, G. R. (eds) Peptides, Chemistry, Structure and Biologyxe2x80x9d, Escom, Leiden (1990), pp. 722-773).
The peptides of the invention can be synthesized by various methods which are known in principle, namely by chemical coupling methods (cf. Wunsch, E: xe2x80x9cMethoden der organischen Chemietxe2x80x9d, Volume 15, Band 1+2, Synthese von Peptiden, thime Verlag, Stutt (1974), and Barrany, G.; Marrifield, R. B.: xe2x80x9cThe Peptidesxe2x80x9d, eds. E. Gross, J. Meienhofer, Volume 2, Chapter 1, pp. 1-284, Academic Press (1980)), or by enzymatic coupling methods (cf. Widmer, F. Johansen, J. T., Carlsberg Res. Commun., Vol.44, pp. 37-46 (1979), and Kullmann, W.: xe2x80x9cEnzymatic Peptide Synthesisxe2x80x9d, CRC Press Inc. Boca Raton, Fla. (1987), and Widmer, F., Johansen, J. T. in xe2x80x9cSynthetic Peptides in Biology and Medicinesxe2x80x9d, eds. Alitalo, K., Partanen, P., Vatieri, A., pp.79-86, Elsevier, Amsterdam (1985)), or by a combination of chemical and enzymatic methods if this is advantageous for the process design and economy.
A cysteine residue may be added at both the amino and carboxy terminals of the peptide, which will allow the cyclization of the peptide by the formation of a disulphide bond.
Any modifications to the peptides of the present invention which do not result in a decrease in biological activity are within the scope of the present invention.
There are numerous examples which illustrate the ability of anti-idiotypic antibodies (anti-Id Abs) to an antigen to function like that antigen in its interaction with animal cells and components of cells. Thus, anti-Id Abs to a peptide hormone antigen can have hormone-like activity and interact specifically with a mediator in the same way as the receptor does. (For a review of these properties see: Gaulton, G. N. and Greane, M. I. 1986. Idiotypic mimicry of biological receptors, Ann. Rev. Immunol. Vol. 4, pp. 253-280; Sege K. and Peterson, P. A., 1978, Use of anti-idiotypic antibodies as cell surface receptor probes, Proc. Natl. Acad. Sci. U.S.A., Vol. 75, pp. 2443-2447).
It is expected from this functional similarity of anti-Id Ab and antigen, that anti-Id Abs bearing the internal image of an antigen can induce immunity to such an antigen. (See review in Hiernaux, J. R., 1988, Idiotypic vaccines and infectious diseases, Infect. Immun., Vol. 56, pp. 1407-1413).
It is, therefore, possible to produce anti-idiotypic antibodies to the peptides of the present invention which will have similar biological activity.
Accordingly, the present invention also provides anti-idiotypic antibodies to the peptides of the present invention, the anti-idiotypic antibody being capable of inhibiting TNF toxicity, but not its binding to the receptor.
The individual specificity of antibodies resides in the structures of the peptide loops making up the Complementary Determining Regions (CDRs) of the variable domains of the antibodies. Since in general the amino acid sequence or the CDR peptides of an anti-Id Ab are not identical to or even similar to the amino acid sequence of the peptide antigen from which it was originally derived, it follows that peptides whose amino acid sequence in quite dissimilar, in certain contexts, can take up a very similar three-dimensional structure. The concept of this type of peptide, termed a xe2x80x9cfunctionally equivalent sequencexe2x80x9d or mimotope by Geyson is known. (Geyson, H. M. et al, 1987, Strategies for epitope analysis using peptide synthesis., J. Immun. Methods, Vol. 102, pp. 259-274).
Moreover, the three-dimensional structure and function of the biologically active peptides can be simulated by other compounds, some not even peptidic in nature, but which nevertheless mimic the activity of such peptides. This field is summarized in a review by Goodman, M. (1990), (Synthesis, Spectroscopy and computer simulations in peptide research, Proc. 11th American Peptide Symposium published in Peptides-Chemistry Structure and Biology, pp. 3-29; Eds. Rivier, J. E. and Marshall, G. R. Publisher Escom).
It is also possible to produce peptide and non-peptide compounds having the same three-dimensional structure as the peptides of the present invention. These xe2x80x9cfunctionally equivalent structuresxe2x80x9d or xe2x80x9cpeptide mimicsxe2x80x9d will react with antibodies raised against the peptide of the present invention and may also be capable of inhibiting TNF toxicity.
Accordingly, a further embodiment of the present invention provides a compound the three-dimensional structure of which is similar as a pharmacophore to the three-dimensional structure of the peptides of the present invention, the compound being characterized in that it reacts with antibodies raised against the peptides of the present invention and that the compound is capable of inhibiting TNF toxicity.
More detail regarding pharmacophores can be found in Bolin et al., p. 150, Polinsky et al., p. 287, and Smith et al., p. 485, in Smith and Rivier (eds.) xe2x80x9cPeptides: Chemistry and Biologyxe2x80x9d, Escom, Leiden (1991).
All of the molecules (proteins, peptides, etc.) may be produced either by conventional chemical methods, as described herein, or by recombinant DNA methods.
All of the molecules (proteins, peptides, etc.) may be produced either by conventional chemical methods, as described herein, or by recombinant DNA methods.
The invention also provides DNA molecules encoding the ligands according to the invention, vectors containing them and host cells comprising the vectors and capable of expressing the ligands according to the invention.
The host cell may be either prokaryotic or eukaryotic.
The invention further provides DNA molecules hybridizing to the above DNA molecules and encoding ligands having the same activity.
The invention also provides pharmaceutical compositions comprising the above ligands which are useful for treating diseases induced or caused by the effects of TNF, either endogenously produced or exogenously administered.
The invention also provides for using the ligands according to the invention for increasing the inhibitory effect of a soluble receptor of the TNF/NGF receptor family. As stated above, the soluble receptors, especially those of TNF, have the ability to block the function of TNF by binding it in competition with the surface receptors. Application of a ligand according to the invention together with a soluble receptor is, therefore, expected to increase the inhibitory effect of the soluble receptor.